2A: We asked the question: what are the changes in tyrosine phosphorylation of proteins upon EGF stimulation and tyrosine kinase inhibitor (TKI) inhibition in human lung adenocarcinoma cell lines expressing the mutant EGFRs? We used two TKIs, erlotinib, a reversible EGFR inhibitor and BIBW2992, an irreversible EGFR and ERBB2 inhibitor. Various large-scale experiments were performed using SILAC and mass spectrometry. Lung adenocarcinoma cell lines used in these experiments were H3255 and 11-18 (L858R mutation), H1975 (L858R/T790M mutation), PC9 (E746-A750 Del EGFR). In addition isogenic NR6 (a variant of 3T3 fibroblasts) and HBECs (human bronchial epithelial cells) with stable expression of WT EGFR, L858R EGFR, Del EGFR were also used for phosphorylation studies.200-700 proteins have been identified from each of the above experiments and their relative quantitation performed. Currently we are validating the change in phosphorylation of a subset of these proteins by immunoprecipitation and western blot experiments. We also performed siRNA-mediated knockdown of proteins identified as phosphorylation targets of mutant EGFRs in lung adenocarcinoma cells harboring mutant EGFRs or mutant KRAS. It is interesting to note that several of the proteins whose tyrosine phosphorylation was inhibited by TKIs in mutant EGFR-expressing cells were also required for survival of EGFR mutant expressing cells but not KRAS mutant expressing cells. We are currently following two such targets: DAPP1 and SCAMP3. DAPPI1 (dual adapter for phosphotyrosine or 3-phosphoinositides) has not been implicated in EGFR signaling. However tyrosine phosphorylation at Y139 of DAPPI1 is stimulated upon EGF stimulation and inhibited 5-10 fold upon treatment with TKIs, erlotinib and BIBW2992, suggesting DAPPI1 may be an integral member of the EGFR signaling pathway. We have shown by biochemical experiments that DAPP1 interacts with EGFR. We have also shown that Y139 is a major phosphorylation site of DAPP1. We have planned experiments to assay whether EGFR is the kinase phosphorylating DAPP1, since there is some evidence that SRC may be the kinase involved. SCAMP3 (secretory career membrane protein) has been shown to interact with EGFR. We have demonstrated that mutant EGFRs phosphorylate specific sites in SCAMP3 more than WT EGFR. SCAMP3 is involved in receptor recycling. We are currently studying the role of SCAMP3 in mutant EGFR trafficking. 2B: Identification of Ser/Thr phosphorylation sites on downstream targets of mutant EGFRs and quantitation of phosphorylation changes upon TKI inhibition resistant to EGFR-directed TKIsWe have completed a series of experiments to identify Ser/Thr phosphorylation sites by SILAC labeling of adenocarcinoma cells and mass spectrometry. We used various fractionation techniques after in-solution tryptic digestion such as strong cation exchange (SCX) or basic reverse phase. Around 30 fractions were then subjected to TiO2 enrichment for phosphopeptide isolation followed by reverse phase liquid chromatography and tandem mass spectrometry using an orbitrap velos mass spectrometer. H3255 lung adenocarcinoma cells harboring the L858R mutation were used in a triple-SILAC experiment. A total of 3,586 phosphosites (3167 phosphoserine, 395 phosphothreonine and 24 phosphotyrosine sites) were identified from this study, which corresponds to 1,434 proteins. There were 278 phosphosites that were activated upon EGF treatment and 358 that were dephosphorylated. Majority of phosphosites (2673 sites) did not show any change on treatment with EGF. This observation is reflective of the existence of very specific signaling cascades that are disrupted on treatment with the drug. We have analyzed this data set using several bioinformatic tools, including IPA and Ariadne pathway studio. Various canonical pathways such as p70S6, IRS, ERK/MAPK, mTOR, PKA, JAK/STAT were enriched in our dataset. We are currently repeating similar global phosphoproteomic experiments with another human lung adenocarcinoma cell line, H1975 that has the acquired resistance mutation, T790M, along with the sensitizing mutation, L858R. This cell line is resistant to EGFR-directed TKI, erlotinib. The comparison of these two experiments will identify potential off-target effects of erlotinib, and also have the potential to identify additional targets to overcome drug resistance.